m-bis(2,3-epoxypropoxy)benzene

Repeated dose toxicity: dermal

Administrative data

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

No Guideline available.

Deviations:

not applicable

Principles of method if other than guideline:

2 year chronic dermal study conducted in Mice.
Animals were treated three times weekly and observed for systemic toxicity.
Full details available under the methods and results section.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

m-bis(2,3-epoxypropoxy)benzene

EC Number:

202-987-5

EC Name:

m-bis(2,3-epoxypropoxy)benzene

Cas Number:

101-90-6

Molecular formula:

C12H14O4

IUPAC Name:

2-({3-[(oxiran-2-yl)methoxy]phenoxy}methyl)oxirane

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Ciba Geige
- Lot/batch No.of test material: P6602
- Purity test date: 88% (wt)
- Impurities:
total chlorine 1%
toluene 6000 ppm
epichlorohydrin 845 ppm
phenyl glycidyl ether 406 ppm
toluene glycidyl ether present
monochlorohydrin <10 ppm
several aromatic species containing oxygen were present

- Storage condition of test material: at 4°C

Test animals

Species:

mouse

Strain:

C3H

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NA
- Age at study initiation: 10 weeks
- Weight at study initiation: NA
- Housing: in polycarbonate shoebox cages with hardwood chip bedding
- Diet: ad libitum Purina 5010-C
- Water: ad libitum

Administration / exposure

Type of coverage:

not specified

Vehicle:

acetone

Details on exposure:

TEST SITE
- Area of exposure: not specified
- % coverage: not specified
- Time intervals for shavings or clipplings: as required

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.45, 0.9 and 1.8 mg/week (or 0.15, 0.3 and 0.6 mg/day/animal)
- Concentration (if solution): 0.32, 0.63 and 1.25% (wt/vol)

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

24 months

Frequency of treatment:

Three days per week: on Monday, Wednesday and Friday

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.45 other: mg/week

Remarks:

exposure: 3 days/week

Doses / concentrations 2

Dose / conc.:

0.9 other: mg/week

Remarks:

exposure: 3 days/week

Doses / concentrations 3

Dose / conc.:

1.25 other: mg/week

Remarks:

exposure: 3 days/week

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The corrosiveness of the test item limited the MTDD to 1.8 mg/week.
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: not specified. Average body weight intervals: 6, 12 and 24 months at the highest dosage

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the experiment
- Anaesthetic used for blood collection: Yes (Metofane (R) of Pitman-Moore)
- Animals fasted: No data
- How many animals: 33
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the experiment
- Animals fasted: No data
- How many animals: 35
- Parameters checked in table 2 were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: yes, but results were not specified in the present report

HISTOPATHOLOGY: yes, but results were not specified in the present report

Statistics:

Body weight was evaluated by the t-test for comparison of treated group means with those of the vehicle control. Group means and standard errors were calculated for clinical hematologic and chemical parameters. The effect of treatment on systemic mortality and the effect of the presence of skin tumor on mortality were evaluated by means of the Mantel-Haenszel test on the force of mortality. The "force of mortality" for an animal alive at the beginning of a small time interval is the probability of death in the interval divided by the length of the interval. Here each interval is taken to be 1 day. The degree of skin carcinogenicity was determined from parameters of the Weibull distribution fitted to the times to tumor for each animal.

Results and discussion

Results of examinations

Clinical signs:

not specified

Dermal irritation:

no effects observed

Description (incidence and severity):

severe skin irritation above 1.25% (or 1.8 mg/day dose) in the dose finding study. However, at doses selected for the long-term study no skin irritation was observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

see Table 3 Overall and systemic mortality in CH3 mice exposed dermally for 24 months to diglycidyl ether of recorcinol and acetone control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see Table 4 Body weight in male and female CH3 mice exposed dermally at the highest dosage to diglycidyl ether of recorcinol and acetone control

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Leukopenia was noted with the test item at the two highest dose levels. Because differential counts were not done, it is not possible to determine whether suppression of lymphocytes, granulocytes, or both, contributed to the decrease in white cell numbers. The fact that red cell numbers were not reduced in animals exposed to the test item makes it least likely that bone marrow toxicity caused the leukopenia. By default, it is more likely that toxicity to lymphatic tissues reduced the cell count.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A clear treatment- and dose-related hypoglycemia was noted in both male and female mice exposed to the test item

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

The repeated test item application to the mouse skin during 24 months (3 exposures/week) confirmed the evidence that diglycidyl ether of resorcinol is a systemic toxin at levels that fail to induce either skin irritation or neoplasia.

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

4.8 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Table 3. Overall and systemic mortality in CH3 mice exposed dermally for 24 months to diglycidyl ether of recorcinol and acetone control

Dose (mg/week)	Sex	% Survival at 750 days (95% confidence limits)	Χ^2for systemic mortality^a
1.8	F	17 (6-36)	66.61***
1.8	M	72 (44-88)	1.47
0.9	F	64 (43-81)	3.93*
0.9	M	72 (53-88)	0.76
0.45	F	84 (66-94)	0.22
0.45	M	84 (66-94)	0.18
Acetone control	F	82 (76-88)	-
Acetone control	M	83 (77-90)	-

*P<0.05, ***P<0.001

Table 4. Body weight in male and female CH3 mice exposed dermally at the highest dosage to diglycidyl ether of recorcinol and acetone control

Dose (mg/week)	Sex	Average body weight (SE) in g at
		6 months	12 months	24 months
1.8	F	25.9 (0.3)	25.5 (0.2)	18 (1)*
1.8	M	31.3 (0.5)	30 (0.3)	22 (0.7)*
Acetone control	F	28.2 (0.5)	27.2 (0.3)	27.4 (0.3)
Acetone control	M	33.4 (0.5)	31.8 (0.5)	30 (0.6)

*Mean body weight significantly different form that of vehicle control; P<0.001

Applicant's summary and conclusion

Conclusions:

Under the conditions tested, the test item showed for systemic toxicity the lowest observed adverse effect (LOAEL) at 0.9 mg/week or 4.8 mg/kg bw/day dose (assuming mouse body weight of 27 g). For the local effects the test item fail to induce either skin irritation or neoplasia at the highest dose tested (1.8 mg/week or 0.257 mg/day or 6.27 µg/cm2, assuming 27 g bw of mouse).

Executive summary:

Inbred, male and female C3Hf/Bd mice were produced under pathogen-free barrier conditions and held under these conditions for the 24-month duration of the experiment. Mice were weaned at 3-4 weeks of age. At 10 weeks of age the hair was removed from the back with electric clippers and the animals were randomly assigned in groups of five to each treatment dose combination. Mice were housed in polycarbonate shoebox cages with hardwood chip bedding. Food (Purina 5010-C) and water were constantly available.

The dose levels for the test item was selected on the basis of a 2-week, five times weekly application of the test material dissolved in spectra-grade acetone (Matheson-Coleman-Bell). The highest dose for the 2-year exposure was one which could be tolerated without irreversible local skin toxicity or systemic toxicity as reflected by suppression of weight gain or mortality. When no significant local or'systemic toxicity was observed, the viscosity of the material determined the concentration which could be reproducibly applied to the animals. At the start of the experiment the test item was weighed into glass scintillation vials in an amount sufficient to yield the highest concentration after the addition of an appropriate volume of acetone. The vials were kept in the dark at 4°C until used.

The material was applied with a 50 µl micropipette on Monday, Wednesday and Friday, excluding holidays. Mice were reshaved as required. The time of neoplasm appearance was taken as the day on which a raised, circumscribed lesion appeared in the treated area of skin that persisted for the duration of the experiment or until death.

At intervals throughout the experiment body weight was determined, by cage group, at the highest concentration of the test item. Cumulative mortality was noted. Heparinized blood samples taken from a random sample of mice surviving the full 24 month exposure were submitted to the clinical laboratory of the ORNL Health Division. For each sample total red and white cell counts, hematocrit and hemoglobin were determined before the sample was centrifuged to recover the plasma. The plasma was subjected to analysis for total protein, albumin, glutamic-oxalacetic transaminase, alkaline phosphatase, urea nitrogen, glucose and triglycerides.  The viscera were examined in animals that either died or were killed at the end of the study. Lesions noted were recorded on a standard form for each animal and tissues were taken for histology only when the gross diagnosis was questionable.

Body weight was evaluated by the t-test for comparison of treated group means with those of the vehicle control. Group means and standard errors were calculated for clinical hematologic and chemical parameters. The effect of treatment on systemic mortality and the effect of the presence of skin tumor on mortality were evaluated by means of the Mantel-Haenszel test on the force of mortality. The "force of mortality" for an animal alive at the beginning of a small time interval is the probability of death in the interval divided by the length of the interval. Here each interval is taken to be 1 day. The degree of skin carcinogenicity was determined from parameters of the Weibull distribution fitted to the times to tumor for each animal.

Evaluation of chronic toxicity was based upon changes in body weight at the highest dose of the test material, changes in force of non-skin tumor mortality and blood hematologic and chemical alterations at the highest dose level of the test material, relative to the vehicle control.

Body weight was significantly (P < 0.001) suppressed at 24 months in male and female mice exposed to the test item. Mortality was summarized in the result table. Animals that were killed before 750 days were ignored in this calculation. Significant (P < 0.05) systemic mortality was noted at the higher doses, especially in female mice.

Toxic suppression of bone marrow and lymphatic tissues was evaluated by examining the cellular composition of peripheral blood in a randomly selected subset of animals which survived to the end of the experiment. Blood was collected by cardiac puncture, under Metofane(R) (Pitman-Moore) anesthesia, into a syringe which contained heparin to prevent coagulation. The samples were evaluated for total red cell count, total white cell count, hemoglobin and hematocrit by use of conventional clinical laboratory procedures. Treatment-related effects were evaluated by comparison of the data with that from acetone controls, while age- (or acetone-) related changes were evaluated by comparison of vehicle control with 10- to 12-week-old untreated mice (aging control). Only the highest dose of each material was evaluated for which body weight suppression and mortality provided evidence of systemic toxicity.

The data indicate an age- (or acetone-) related decrease in the number of circulating red cells and a proportionate decrease in hemoglobin and hematocrit in all treated animals.

Leukopenia was noted with the test item at the two highest dose levels. Because differential counts were not done, it is not possible to determine whether suppression of lymphocytes, granulocytes, or both, contributed to the decrease in white cell numbers. The fact that red cell numbers were not reduced in animals exposed to the test item makes it least likely that bone marrow toxicity caused the leukopenia. By default, it is more likely that toxicity to lymphatic tissues reduced the cell count.

Plasma total protein levels were increased as a function of age in both sexes, with occasionial high average levels noted in males. Alkaline phosphatase levels varied widely, both within and between groups. It appeared that female C3H mice had significantly higher alkaline phosphatase levels than males, irrespective of age. Glutamic-oxalacetic transaminase levels were relatively uniform across groups and between sexes, but the range of standard errors gives a clear indication of underlying individual heterogeneity. Glucose levels were little affected by sex or age. A clear treatment- and dose-related hypoglycemia was noted in both male and female mice exposed to the test item. Triglyceride levels varied widely, with no clear indication of an effect of treatment or age, although levels were consistently higher in males than in females. Urea nitrogen levels were higher in males than females and also increased as a function of age. Treatment-related differences were not apparent, although standard errors again suggested considerable variation among individual animals. Taken together, these data amplify and confirm the evidence that the test material is a systemic toxin at dose levels that fail to-induce either skin irritation or neoplasia. Since clinical hematologic and chemical analyses were conducted on individual animals and each animal was also subjected to gross and microscopic evaluation, it eventually will be possible to correlate clinical findings with gross and microscopic pathologic changes.

Skin neoplasms were not induced by the test item at the dosage levels applied to C3H mice. The test item also attracts attention as a potential systemic toxicant at levels of exposure that do not induce local irritation. Previously published animal carcinogenicity studies of the test item reveal that it is capable of eliciting skin neoplasms in C57BL, but not in Swiss ICR mice following chronic skin exposure. This is consistent with the present data in which skin tumors were not induced in C3H mice, which have previously been shown to be less sensitive to chemical skin carcinogenesis than C57BL/6.

In opinion of the authors, the most significant occupational risk for the test item would be primary skin irritation and potential systemic toxicity associated with skin absorption. It is unlikely that chronic dermal exposures at concentrations above those used in the present experiment would be unnoticed by workers due to the irritant properties of this material.

In conclusion, under the conditions tested, the test item showed the lowest observed adverse effect (LOAEL) at 0.9 mg/week or 4.8 mg/kg bw/day dose (assuming mouse body weight of 27 g).